The topoisomerase II protein (ORF 045L) of invertebrate iridescent virus 6 (IIV6) plays essential roles in managing DNA topology during viral replication and transcription. Considering the importance of the topoisomerase II gene, a comprehensive analysis was conducted to explore the codon usage bias (CUB) of topoisomerase II genes of IIV6 and 9 reference invertebrate iridescent viruses (IIVs). In this research, the findings from the base composition analysis revealed that the IIV6 topoisomerase gene had a high A/T content, with nucleotide A being the most prevalent. The relative synonymous codon usage values for each codon demonstrated the presence of CUB. The effective number of codons (ENC) value for the IIV6 topoisomerase II gene is 34.80, signifying a significant CUB. The ENC plot indicates that all the diverse sequences lie beneath the standard curve, signifying that CUB is influenced not only by mutational pressure but also by other factors, including natural selection. The findings from the neutrality analysis indicate that the codon usage pattern (CUP) is more significantly shaped by natural selection, as evidenced by a regression line slope of 0.1602, compared to the influence of mutation pressure. Furthermore, it has been established that the nucleotide composition and dinucleotide content influence the CUB of the topoisomerase II gene in IIV6. The initial comprehensive analysis of CUB in the IIV6 topoisomerase II gene offers valuable insights into the gene's evolutionary processes.